Wnt/sfrp complexes, wnt-containing compositions, wnt-expressing cells, and methods of making, purifying, and using same

ABSTRACT

This disclosure describes isolated protein complexes including a Wnt and a sFRP; compositions including a Wnt; a cell overexpressing a Wnt and a sFRP; compositions including a cell overexpressing a Wnt and a cell overexpressing a sFRP; methods of making the protein complexes, compositions, and cells; and methods of using the isolated protein complexes, compositions, and cells. This disclosure further describes methods of forming a complex including a Wnt and a sFRP and methods for isolating a Wnt. Also described herein are methods that may be used to purify a Wnt without the use of a detergent.

CONTINUING APPLICATION DATA

This application claims the benefit of U.S. Provisional Application Ser.No. 62/569,748, filed Oct. 9, 2017, which is incorporated by referenceherein.

SEQUENCE LISTING

This application contains a Sequence Listing electronically submitted tothe United States Patent and Trademark Office via EFS-Web as an ASCIItext file entitled “541-00060101_ST25.txt” having a size of 84 kilobytesand created on Oct. 9, 2018. Due to the electronic filing of theSequence Listing, the electronically submitted Sequence Listing servesas both the paper copy required by 37 CFR § 1.821(c) and the CRFrequired by § 1.821(e). The information contained in the SequenceListing is incorporated by reference herein.

SUMMARY OF THE INVENTION

This disclosure describes an isolated protein complex that includes aWingless/Integrated-1 protein (Wnt) and a secreted Frizzled-relatedprotein (sFRP); a composition that includes a Wnt, wherein thecomposition is substantially free of a detergent; a cell overexpressinga Wnt and a sFRP; compositions including a cell overexpressing a Wnt anda cell overexpressing a sFRP; methods of making the protein complexes,compositions, and cells; and methods of using the protein complexes,compositions, and cells. In some embodiments, the Wnt preferablyincludes an active Wnt. In some embodiments, the compositions may beused as a media additive and may provide advantages versus compositionsthat include detergent.

In one aspect, this disclosure describes an isolated protein complexincluding a Wnt and a sFRP.

In another aspect, this disclosure describes a composition including aWnt, wherein the composition is substantially free of a detergent.

In a further aspect, this disclosure describes a cell overexpressing aWnt and a sFRP.

In an additional aspect, this disclosure describes a compositionincluding a cell overexpressing a Wnt and a cell overexpressing a sFRP.

In yet another aspect, this disclosure describes a method includingforming a complex including a Wnt and a sFRP and isolating the Wnt.

The words “preferred” and “preferably” refer to embodiments of theinvention that may afford certain benefits, under certain circumstances.However, other embodiments may also be preferred, under the same orother circumstances. Furthermore, the recitation of one or morepreferred embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the invention.

The terms “comprises” and variations thereof do not have a limitingmeaning where these terms appear in the description and claims. By“consisting of” is meant including, and limited to, whatever follows thephrase “consisting of.” Thus, the phrase “consisting of” indicates thatthe listed elements are required or mandatory, and that no otherelements may be present. By “consisting essentially of” is meantincluding any elements listed after the phrase, and limited to otherelements that do not interfere with or contribute to the activity oraction specified in the disclosure for the listed elements. Thus, thephrase “consisting essentially of” indicates that the listed elementsare required or mandatory, but that other elements are optional and mayor may not be present depending upon whether or not they materiallyaffect the activity or action of the listed elements.

Unless otherwise specified, “a,” “an,” “the,” and “at least one” areused interchangeably and mean one or more than one.

Also herein, the recitations of numerical ranges by endpoints includeall numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.80, 4, 5, etc.).

For any method disclosed herein that includes discrete steps, the stepsmay be conducted in any feasible order. And, as appropriate, anycombination of two or more steps may be conducted simultaneously.

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The description that follows more particularly exemplifiesillustrative embodiments. In several places throughout the application,guidance is provided through lists of examples, which examples may beused in various combinations. In each instance, the recited list servesonly as a representative group and should not be interpreted as anexclusive list.

Reference throughout this specification to “one embodiment,” “anembodiment,” “certain embodiments,” or “some embodiments,” etc., meansthat a particular feature, configuration, composition, or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the disclosure. Thus, the appearances of such phrases invarious places throughout this specification are not necessarilyreferring to the same embodiment of the disclosure. Furthermore, theparticular features, configurations, compositions, or characteristicsmay be combined in any suitable manner in one or more embodiments.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless otherwise indicated to thecontrary, the numerical parameters set forth in the specification andclaims are approximations that may vary depending upon the desiredproperties sought to be obtained by the present invention. At the veryleast, and not as an attempt to limit the doctrine of equivalents to thescope of the claims, each numerical parameter should at least beconstrued in light of the number of reported significant digits and byapplying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. All numerical values, however, inherently contain a rangenecessarily resulting from the standard deviation found in theirrespective testing measurements.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1(A-B) shows co-expression of mouse sFRP1 (msFRP1) and mouse Wnt1(mWnt1) in CHO cells results in higher levels of active mWnt1 protein inthe supernatant (also referred to herein as conditioned media). FIG. 1A.Western blots of equal amounts of conditioned media (CM) shows enhancedlevels of mWnt1 in CHO cells expressing mWnt1/msFRP1-His (lane 2) andmWnt1/msFRP1 (lane 5) compared to mWnt1 levels in CHO control cell CM(lane 1), CHO mWnt1 CM (lane 3), and CHO msFRP1 (lane 4). msFRP1 Westernblots show enhanced msFRP1 expression in CHO mWnt1/msFRP1-His CM (lane2), CHO msFRP1 CM (lane 4), and CHO mWnt1/msFRP1 CM (lane 5) compared toCHO CM (lane 1) and CHO mWnt1 CM (lane 3). FIG. 1B. Conditioned mediafrom CHO cells (blue circles), CHO mWnt1 (red squares), CHO mWnt1/msFRP1(green triangles), CHO mWnt1/msFRP1-His (orange diamonds) and CHO msFRP1(red circles) were added to HEK293 TCF9-SEAP hFz4/hLRP5 Wnt Reportercells starting at the highest concentration of media and diluting 1:2.Only when CHO cells expressed mWnt1 and msFRP1 or msFRP1-His togetherwas Wnt1 activity detected in the conditioned media. The conditionedmedia from CHO-s mWnt1/msFRP1 cells (green triangles) resulted in a22-fold induction while the CHO mWnt1/msFRP-His (orange diamonds)resulted in a 17-fold induction above background in this HEK293TCF9-SEAP hFz4/hLRP5 Wnt Reporter assay. Conditioned media from theother CHO lines did not induce activity in the HEK293 TCF9-SEAPhFz4/hLRP5 Wnt Reporter cell line.

FIG. 2A shows msFRP1-His binds to mWnt1 in CHO conditioned mediaexpressing both mWnt1 and msFRP1-His. Immunoprecipitation experimentswere performed by adding anti-Wnt1 antibody (lane 2, lane 4), anti-Hisantibody (lane 3, lane 5), or no antibody control (lane 1, lane 6) toequal amounts of CHO cell conditioned media overexpressing both mWnt1and msFRP1-His. The antibody/conditioned media mixtures were complexedto protein G agarose beads and immunoprecipitation experiments wereperformed. When no antibody was added to the CHO mWnt1/msFRP/Hisexpressing conditioned media, msFRP1 and mWnt1 were not detected inWestern blots (lanes 1 and 6 respectively). When an anti-mWnt1 antibodywas used to immunoprecipitate, the His tagged msFRP1/His protein wasdetected at around 37 kilodaltons (kDa) when blotted with an anti-Hisantibody (lane 2). When an anti-His antibody was used toimmunoprecipitate, the anti-mWnt1 antibody detected a mWnt1 band ataround 42 kDa (lane 5). The bands at 50 kDa and 25 kDa in lane 3 areheavy and light chain IgGs being recognized by the anti-His antibody.FIG. 2B shows msFRP1 binds to mWnt2b in CHO conditioned media expressingboth mWnt2b and msFRP1. Immunoprecipitation experiments were performedby adding anti-mouse Wnt2b, anti-human sFRP1, or no antibody control toequal amounts of CHO cell conditioned media overexpressing mWnt2b aloneor mWnt2b and msFRP1 (Clone 18). Immunoprecipitation with anti-mWnt2bantibodies followed by blotting with anti-hsFRP1 antibodies resulted inthe detection of sFRP1 protein at around 35 kDa (indicated by blackarrows), demonstrating that mWnt2b and msFRP1 are physically interactingin the conditioned media of CHO cells expressing both mWnt2b and msFRP1.This 35 kDa band was not detected in the conditioned media of CHO cellsexpressing only mWnt1 when immunoprecipitations were performed withanti-mWnt2b and blotted with anti-hsFRP1. FIG. 2C shows msFRP1 binds tomWnt2b in CHO conditioned media expressing both mWnt2b and msFRP1.Immunoprecipitation experiments were performed by adding anti-Wnt2b,anti-hsFRP1, or no antibody control to equal amounts of CHO cellconditioned media overexpressing mWnt2b alone or mWnt2b and msFRP1(Clone 18). Immunoprecipitation with anti-hsFRP1 antibodies followed byblotting with anti-mWnt2b antibodies resulted in the detection of mWnt2bprotein at around 42 kDa (indicated by black arrow), demonstrating thatmWnt2b and msFRP1 are physically interacting in the conditioned media ofCHO cells expressing both mWnt2b and msFRP1. This 42 kDa mWnt1 band wasnot detected in the conditioned media of CHO cells expressing only mWnt1when immunoprecipitations were performed with anti-hsFRP1 and blottedwith anti-mWnt1.

FIG. 3 shows mouse sFRP1 (msFRP1) both enhances and inhibits mouse Wnt1(mWnt1) activity. Recombinant msFRP1 protein was added to CHOmWnt1-expressing cells for 24 hours prior to adding the conditionedmedia to HEK293 hFz4/hLRP5 Wnt reporter cells. sFRP1 protein showed amaximal enhancement of mWnt1 activity at 6.25 micrograms per milliliter(μg/mL) of protein added with doses of 25 μg/mL and 50 μg/mL resultingin reduction of the mWnt1 activity down to baseline levels.

FIG. 4(A-F) shows Wnt-1 co-eluted with sFRP1 on a cation exchanger SPSepharose column (FIG. 4(A-C)) and separation of sFRP1/Wnt-1 complexfrom free sFRP1 on a gel filtration column (FIG. 4(D-E)). FIG. 4A.Conditioned media from CHO cells co-expressing msFRP1 and mWnt-1 wereloaded onto SP Sepharose column, and bound proteins were eluted with alinear gradient of high salt buffer. The distinct late elution peak wasthe position that sFRP was usually eluted. FIG. 4B. Fractions collectedfrom SP elution were loaded onto 15 percent (%) SDS-PAGE and stainedwith silver. The 37 kDa bands represent sFRP1. FIG. 4C. The samefractions of FIG. 4B were subjected to Western blot probed with antibodyagainst mWnt-1. mWnt-1 was detected as a ˜52 kDa band that is present inthe same fractions where sFRP1 was detected. FIG. 4D. Pool from a SPSepharose column was loaded and eluted from a gel filtration (Superdex200) column. The earlier-eluted peak contained the sFRP1/Wnt-1 complex,while the later-eluted peak contained the free sFRP. FIG. 4E. Fractionscollected from gel filtration elution were loaded onto a 15% SDS-PAGEgel and stained with Coomassie blue. The lower band (37 kDa) representssFRP1 and the upper band (52 kDa) represents Wnt-1. FIG. 4F. sFRP1/Wnt-1complex purified from a gel filtration column was loaded onto a 15%SDS-PAGE gel and stained with Coomassie blue. Two bands, at 37 kDa and52 kDa, were observed, with a roughly 1:1 molar ratio.

FIG. 5 shows both mWnt1 and msFRP1 Proteins were detected by WesternBlot when the purified recombinant mWnt1/msFRP1 complex was run on anSDS-PAGE gel. 0.001 nanograms per milliliter (ng/mL) of mWnt1/msFRP1(lane 1), 0.01 ng/mL of mWnt1/msFRP1 (lane 2), 0.1 ng/mL of mWnt1/msFRP1(lane 3), 1 ng/mL of mWnt1/msFRP1 (lane 4), 10 ng/mL of mWnt1/msFRP1(lane 5), 100 ng/mL of mWnt1/msFRP1 (lane 6), 500 ng/mL of mWnt1/msFRP1(lane 7), and 1 microgram per milliliter (μg/mL) of mWnt1/msFRP1 (lane8) were run on a 4-20% acrylamide gel under reducing conditions and thentransferred to PVDF membrane. Western Blotting was performed with a goatanti-Wnt-1 antibody (1 μg/mL) and a goat anti-msFRP1 antibody (1 μg/mL).A secondary antibody (donkey anti-goat HRP antibody) was used at 1 μg/mLfor detection.

FIG. 6(A-D) shows a purified recombinant mWnt1/msFRP1 protein complexactivates the Wnt signaling pathway in HEK293 Wnt reporter cellsexpressing both hFz4 and hLRP5. FIG. 6A, A mWnt1/msFRP1 protein complexwas tested in HEK293 Wnt reporter cells expressing hFz4/Hlrp5 and shownto be more active compared to recombinant mouse Wnt10B protein. FIG. 6B.HEK293 Wnt Reporter cells expressing human Fz4 and human LRP5 weretreated with either recombinant mouse sFRP1 protein (blue squares) orrecombinant mouse Wnt1/sFRP1 (mWnt1/msFRP1) protein complex (redcircles). The mWnt1/msFRP1 complex demonstrated clear induction of theHEK293 Wnt reporter cells in a dose responsive fashion, while the msFRP1protein did not demonstrate activity alone. FIG. 6C. RecombinantmWnt1/msFRP1 protein complex purification is reproducible with multiplelots showing similar biological activity. Two different mWnt1/msFRP1complexes were purified and compared against each other in the HEK293hFz4/hLRP5 Wnt reporter assay and shown to both show similar activity.The maximal enhancement of Wnt reporter activity was detected at 110ng/mL of the complexes; the activity of these mWnt1/msFRP1 complexescreated a bell shaped curve with doses less than 110 ng/mL or greaterthan 110 ng/mL showing lower activity. FIG. 6D. Mouse Wnt1/sFRP1complexes, purified as described in FIG. 4, are highly potent anddemonstrate better potency in HEK293 Wnt reporter assays compared to themost active Wnt (recombinant mouse Wnt3a) purified and stored in CHAPSbuffer. The observed effective dose of 50 percent (ED₅₀) formWnt1/msFRP1 was 1.8 ng/mL while the observed ED₅₀ for Recombinant mouseWnt3a was 12.2 ng/mL.

FIG. 7(A-C) shows sFRPs enhance exemplary Wnt (Wnt1, Wnt2b, and Wnt6)activity in a cell non-autonomous fashion. FIG. 7A. Conditioned mediafrom cells expressing msFRP1, msFRP3, msFRP4, and msFRP5 was added toHEK293 Wnt reporter cells and did not induce Wnt reporter activity aboveHEK293 Wnt reporter activity alone. When cells expressing mWnt1 werecultured together with cells expressing msFRP1 and msFRP5 for 24 hours,this co-cultured conditioned media induced Wnt pathway activity whenadded to HEK293 Wnt reporter cells. FIG. 7B. Similar experiments showthat conditioned media from co-cultures of cells expressing mouse Wnt2balong with msFRP1 also resulted in Wnt pathway activation. FIG. 7C.Conditioned media from human Wnt6 expressing cells co-cultured withmsFRP1 or msFRP5 expressing cells also resulted in a significantincrease in Wnt reporter activity.

FIG. 8 shows that secreted Wnt proteins (e.g., Wnt3a) bind sFRPhomologs, indicating that secreted Wnt proteins may, like tethered Wnts(e.g., Wnt1, Wnt2b and Wnt6), also be overexpressed with sFRP proteinsand purified in an active state without (3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate) (CHAPS). Purified recombinant mousesFRP proteins were tested in ELISA binding assays with a biotinylatedmouse Wnt3a protein. (Because different tags were used for the differentsFRP proteins, the relative binding affinity cannot be compared usingthese data.) The msFRP1 and msFRP4 were detected with goat anti-sFRP1and sheep anti-sFRP4 antibody, respectively. His tagged-mouse sFRP2 andHis tagged-mouse sFRP3 were detected with a mouse anti-His antibody. HAtagged-mouse sFRP5 was detected with a mouse anti-HA antibody.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

This disclosure describes an isolated protein complex that includes aWingless/Integrated-1 protein (Wnt) and a secreted Frizzled-relatedprotein (sFRP); a composition that includes a Wnt, wherein thecomposition is substantially free of a detergent; a cell overexpressinga Wnt and a sFRP; compositions including a cell overexpressing a Wnt anda cell overexpressing a sFRP; methods of making the protein complexes,compositions, and cells; and methods of using the protein complexes,compositions, and cells. This disclosure further describes methods offorming a complex including a Wnt and a sFRP and isolating the Wnt. Alsodescribed herein are methods that may be used to purify a Wnt including,for example, a tethered Wnt, that could not be purified using Wntpurification protocols available at the time of the invention. Thisdisclosure further describes methods that may be used to purify a Wnt,including, for example, a secreted Wnt, without the use of a detergent.

Wnt proteins are glycosylated and palmitoylated proteins that haveproven extremely difficult to purify in an active state (Willert et al.Cold Spring Harbor Perspectives In Biology, 4:a007864 (2012)). A Wnt mayinclude at least one of Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a,Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b,Wnt11, and Wnt16.

Wnt family members are involved in regulating embryogenesis and controldiverse processes later in life, including cell proliferation, survival,migration, polarity, specification of cell fate, and self-renewal instem cells. Perturbation of the levels of Wnts, or altered activity ofdownstream effectors of Wnts may result in developmental defects and maycontribute to disease etiology.

Due to the lipid modification of Wnt proteins, these growth factors mayassociate with the outer membrane of a Wnt-producing cell, resulting inlimited secretion and signaling away from the Wnt-production source. In2003, a procedure to purify palmitoylated Wnt proteins using(3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate) (CHAPS)detergent was published; for this procedure, maintaining Wnts in ahydrophobic environment proved essential for retaining Wnt activity(Willert et al. Nature 423:448-452 (2003); Reya et al. Nature423:409-414 (2003)). However, a Wnt3a protein purified with CHAPSrapidly loses activity in cell culture media due to its hydrophobicnature, and the presence of the detergent may interfere with normal cellfunction including, for example, stem cell self-renewal, or may provetoxic in cell culture (Tuysuz et al. Nature Communications 8:14578(2017)).

Some Wnts can be detected in an active state in conditioned media (alsoreferred to herein as the supernatant) of Chinese Hamster Ovary (CHO)cells overexpressing Wnts including, for example, Wnt3a, Wnt5a, Wnt5b,Wnt8a, and Wnt10a. These “secreted Wnts” are usually amenable topurification using the aforementioned CHAPS purification protocol. OtherWnts, or “tethered Wnts,” including, for example, Wnt1, Wnt2b, Wnt6,Wnt7a, seem to be primarily associated with the cell membrane of cellsproducing these Wnts. Active Wnt protein is not detected in theconditioned media of cells making these tethered Wnts. The conditionedmedia from cells overexpressing tethered Wnts does not activate Wntsignaling in a paracrine manner, and only when cells making tetheredWnts are co-cultured with Wnt-responsive cells can Wnt activity berealized. Efforts to purify membrane associated proteins have provedextremely difficult, resulting in either purification of inactiveproteins or low production yields that make the product economicallyunviable.

Secreted Frizzled-related proteins (sFRPs) are a family of secreted Wntbinding proteins that were originally described as antagonists of Wntsignaling. There are 5 sFRPs in the human and mouse genomes. sFRP1-5 allshow structural similarity to the Wnt binding cysteine rich domain (CRD)of Frizzled 7-pass transmembrane receptors. Unlike 7-pass transmembraneFrizzled receptors, sFRPs are secreted proteins that contain anetrin-like domain downstream of the CRD and this netrin-like domain isnot present in Frizzled receptors.

Although sFRPs were originally described as secreted proteins that bindto and inhibit Wnt activity, subsequent studies demonstrated that sFRPsmay also enhance Wnt activity including, for example, when expressed atbiologically relevant levels (Mii and Taira, Development 136:4083-4088(2009); Holly et al., Developmental Biology 388:192-204 (2014).

Described herein are experimental results showing that a sFRP expressedin a Wnt-producing cell can bind to and liberate a tethered Wnt from thecell surface. Overexpression of sFRP1 or sFRP5 in Wnt1-expressing cellsresults in detection of active Wnt1/sFRP complexes in the conditionedmedia. This Wnt1/sFRP1 complex can be subsequently purified in anaqueous purification procedure that does not require the use ofdetergents, like CHAPS.

This disclosure also provides experimental results demonstrating thatsFRP-expressing cells, or sFRP recombinant proteins, can liberate anactive Wnt1/sFRP complex from the membrane of Wnt1 expressing cells inboth a cell autonomous and non-cell autonomous fashion. For example, thedata summarized in Table 1 suggest mouse sFRP1 (msFRP1) may bind to andliberate mouse Wnt 1 (mWnt1) from the cell membrane. Only when thesecreted proteins sFRP1 or sFRP5 have contact with mWnt1 on the surfaceof a mWnt1 expressing cell can mWnt1 activity be detected in conditionedmedia. Thus, sFRPs binding to Wnts at the cell surface may allow a Wntprotein to act as a “secreted” morphogen.

Experimental results provided herein also show that sFRPs can alsoliberate other Wnts (including, for example, Wnt2b and Wnt6) from theouter plasma membrane of Wnt-expressing cells, resulting in Wnt2b/sFRPand Wnt6/sFRP complexes in the conditioned media of cells expressingWnt2b or Wnt6. These data demonstrate that a sFRP can act as a Wntbinding partner. However, a biphasic response of sFRPs in the context ofWnt signaling was observed in, for example, FIG. 3, FIG. 6C.

Without wishing to be bound by theory, it is believed that a sFRP maybind to the palmitoylated moiety on a Wnt, shielding this lipidmodification from the aqueous environment and, therefore, allowingtethered Wnts to be purified in an active Wnt/sFRP complex. Again,without wishing to be bound by theory, it is believed that sFRPs bind toand enhance Wnt signaling up to an amount of sFRP that saturates Wntbinding (see FIG. 3, FIG. 6C). However, once all of the available Wntprotein is complexed with a sFRP, sFRP proteins may inhibit Wntsignaling by interacting with other non-Wnt components of the Wntpathway including, for example, Frizzled receptors (Bafico et al. TheJournal of Biological Chemistry 274, 16180-16187 (1999)). For example,excess sFRP may inhibit Wnt proteins from activating Frizzled receptors.

Wnt

A Wnt may include any Wnt protein or combination of Wnt proteins. A Wntprotein may include at least one of Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a,Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b,Wnt10a, Wnt10b, Wnt11, and Wnt16. In some embodiments, the Wnt includesa secreted Wnt and/or a tethered Wnt. In some embodiments, a secretedWnt may include at least one of Wnt3a, Wnt5a, Wnt5b, Wnt8a, and Wnt10a.

In some embodiments, the Wnt includes a eukaryotic Wnt. The Wnt may be aWnt from any suitable eukaryote including, for example, a mammal, anamphibian (e.g., Xenopus), a bird (e.g., a chicken), or a fish (e.g., azebrafish). In some embodiments, the Wnt preferably includes a mammalianWnt. A mammalian Wnt may include, for example, a human Wnt, a mouse Wnt,a rat Wnt, an equine Wnt, a canine Wnt, a caprine Wnt, a bovine Wnt,etc.

In some embodiments, the Wnt includes a tagged Wnt. The Wnt may betagged with any suitable protein tag including, for example, a histidine(His) tag, a FLAG tag, a hemagglutinin (HA) tag, an Fc tag, aglutathione S-transferase (GST) tag, a fluorescent tag (including, forexample, a GFP tag, a YFP tag, a BFP tag), etc. In some embodiments, thetag may be C-terminal; in some embodiments, the tag may be N-terminal.

In some embodiments, the Wnt includes an active Wnt. As used herein, an“active Wnt” is a Wnt that activates canonical β-catenin signaling.

sFRP

A sFRP may include any sFRP protein or combination of sFRP proteins. AsFRP may include, for example, at least one of sFRP1, sFRP2, sFRP3,sFRP4, and sFRP5.

In some embodiments, the sFRP includes a eukaryotic sFRP. The sFRP maybe a sFRP from any suitable eukaryote including, for example, a mammal,an amphibian (e.g., Xenopus), a bird (e.g., a chicken), or a fish (e.g.,a zebrafish). In some embodiments, the sFRP preferably includes amammalian sFRP. A mammalian sFRP may include, for example, a human sFRP,a mouse sFRP, a rat sFRP, an equine sFRP, a canine sFRP, a caprine sFRP,a bovine sFRP, etc.

In some embodiments, the sFRP includes a tagged sFRP. The sFRP may betagged with any suitable protein tag including, for example, a histidine(His) tag, a FLAG tag, a hemagglutinin (HA) tag, an Fc tag, aglutathione S-transferase (GST) tag, a fluorescent tag (including, forexample, a GFP tag, a YFP tag, a BFP tag), etc. In some embodiments, thetag may be C-terminal; in some embodiments, the tag may be N-terminal.

Isolated Protein Complexes and Compositions

In some aspects, this disclosure describes an isolated protein complexincluding a Wnt and a sFRP. In some embodiments, an “isolated proteincomplex” means a protein complex present in a composition or environmentthat is different from that found in nature, that is, from that found ina native or original cellular or body environment. An “isolated proteincomplex” may be separated from at least 50%, at least 75%, at least 90%,or at least 95% of other naturally co-existing cellular or tissuecomponents. An “isolated protein complex” may include a naturallyexisting protein complex in an artificial preparation and/or in anon-native host cell. In some embodiments, an “isolated Wnt/sFRP proteincomplex” may include a Wnt/sFRP complex isolated from a cell engineeredto express unnaturally high levels of recombinant Wnt and sFRP. In someembodiments, the cell may include a non-native host cell and/or the cellmay include non-native DNA integrated into the genome of the cell.

In some embodiments, the isolated protein complex is preferablysubstantially free of a detergent. An isolated protein complex that is“substantially free of” a detergent does not include enough detergent tomaterially affect the activity or action of the Wnt in the isolatedprotein complex. In some embodiments “substantially free of a detergent”means containing less than 2% weight/volume (w/v), less than 1% (w/v),less than 0.5% (w/v), less than 0.1% (w/v), or less than 0.01% (w/v) ofa detergent. In some embodiments, the detergent includes a zwitterionicand/or amphoteric detergent. In some embodiments, the detergent includesat least one of CHAPS (also referred to as3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), CHAPSO (alsoreferred to as3-([3-Cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate),Triton-X-100, Tween 20, Tween 80, SDS, deoxycholate, cholate, sarkosyl,DDM, digitonin, and urea. In some embodiments, the detergent includesCHAPS.

In another aspect, this disclosure describes a composition including aWnt. The composition is preferably substantially free of a detergent. Acomposition that is “substantially free of” a detergent does not includeenough detergent to materially affect the activity or action of the Wntin the composition.

In some embodiments, the Wnt of the isolated protein complex orcomposition preferably includes active Wnt. As used herein, an “activeWnt” is a Wnt that activates canonical β-catenin signaling.

Canonical β-catenin signaling may be measured using any method known toa person having skill in the art. For example, canonical β-cateninsignaling may be measured by assaying for Wnt mediated phosphorylationof β-catenin, LRP5, LRP6, GSK3B, Axin, and/or Dishevelled.Phosphorylation may be measured using Western blot. Autocrine andparacrine C57MG cells, cell transformation assays, and duplication ofthe embryonic axis in Xenopus laevis, or zebrafish, are also establishedassays to test for Wnt-mediated canonical β-catenin activity. (See,e.g., Shimizu et al., Cell Growth Differ. 1997, 8(12):1349-58; Wong etal., Mol. Cell. Biol. 1994, 14(9):6278-86; McMahon and Moon, Cell. 1989,58(6):1075-1084; Lustig et al., Mol. Cell. Biol. 2002, 22(4):1184-93;Tamai et al., Mol. Cell. 2004, 13(1):149-156; Van Noort et al., J BiolChem. 2002, 277(20):17901-17905; Molenaar et al., Cell. 1996,86(3):391-399; Klein and Melton, Proc Natl Acad Sci USA 1996,93(16):8455-8459.)

In some embodiments, canonical β-catenin signaling may be measured usinga secreted alkaline phosphatase (SEAP) reporter assay including theHEK293 TCF9-SEAP hFz4/hLRP5 Wnt Reporter assay described in Example 1.In some embodiments, a protein complex including an active Wnt has a2-fold or greater Wnt reporter activity than a complex not including aWnt, as measured using the SEAP reporter assay. In some embodiments, acomposition including an active Wnt has a 2-fold or greater Wnt reporteractivity than a composition not including a Wnt, as measured using theSEAP reporter assay.

In some embodiments, an active Wnt or an isolated protein complexexhibits an effective dose of 50 percent (ED₅₀) of less than 1000 ng/mL,less than 500 ng/mL, less than 100 ng/mL, less than 50 ng/mL, less than40 ng/mL, less than 30 ng/mL, less than 20 ng/mL, less than 15 ng/mL,less than 10 ng/mL, less than 8 ng/mL, less than 5 ng/mL, less than 4ng/mL, less than 3 ng/mL, or less than 2 ng/mL, as measured using, forexample, a HEK293 TCF9-SEAP hFz4/hLRP5 Wnt Reporter assay, as describedherein.

In some embodiments, an active Wnt or an isolated protein complexexhibits an effective dose of 50 percent (ED₅₀) of at least 2 ng/mL, atleast 3 ng/mL, at least 4 ng/mL, at least 5 ng/mL, at least 8 ng/mL, atlast 10 ng/mL, at least 15 ng/mL, at least 20 ng/mL, at least 30 ng/mL,at least 40 ng/mL, at least 50 ng/mL, at least 100 ng/mL, or at least500 ng/mL, as measured using, for example, a HEK293 TCF9-SEAP hFz4/hLRP5Wnt Reporter assay, as described herein.

In some embodiments, the isolated protein complex or composition mayinclude a Wnt-sFRP fusion protein. In some embodiments, the Wnt may befused to the sFRP1 via a linker including, for example, a peptidelinker. Any suitable linker may be used. In some embodiments, a peptidelinker may include at least one of GGGS (SEQ ID NO:1), GGGGS (SEQ IDNO:2), GSGSG (SEQ ID NO:3), GGGGG (SEQ ID NO:4), and GSGSGGSGSG (SEQ IDNO:5). In some embodiments, the peptide linker may include multiplerepeats of one or more of the peptide linkers described above including,for example, (GGGS)_(X) (SEQ ID NO:6), (GGGGS)_(X) (SEQ ID NO:7);(GSGSG)_(X) (SEQ ID NO:8), (GSGSGGSGSG)_(X) (SEQ ID NO:9), and/or G_(X),where X=1-400.

In some embodiments, the isolated protein complex or composition mayinclude a member of the R-Spondin family including, for example, atleast one of R-Spondin 1, R-Spondin 2, R-Spondin 3, and R-Spondin 4.

In some embodiments, the R-Spondin may be fused to the sFRP1 and/or theWnt, forming a fusion protein. In some embodiments, the R-Spondin may befused to the sFRP1 and/or the Wnt via a linker including, for example, apeptide linker. Any suitable linker may be used. In some embodiments, apeptide linker may include at least one of GGGS (SEQ ID NO:1), GGGGS(SEQ ID NO:2), GSGSG (SEQ ID NO:3), GGGGG (SEQ ID NO:4), and GSGSGGSGSG(SEQ ID NO:5). In some embodiments, the peptide linker may includemultiple repeats of one or more of the peptide linkers described aboveincluding, for example, (GGGS)_(X) (SEQ ID NO:6), (GGGGS)_(X) (SEQ IDNO:7); (GSGSG)_(X) (SEQ ID NO:8), (GSGSGGSGSG)_(X) (SEQ ID NO:9), and/orG_(X), where X=1-400.

In some embodiments, the isolated protein complex or composition mayinclude Lipocalin7. In some embodiments, the isolated protein complex orcomposition may include WIF1.

Methods of Making

In a further aspect, this disclosure describes a method that includesforming a complex including a Wnt and a sFRP and isolating the Wnt. Insome embodiments, the Wnt may be isolated before forming a complexincluding a Wnt and a sFRP. In some embodiments, the Wnt may be isolatedfrom a complex including a Wnt and a sFRP. In some embodiments,including when the Wnt is isolated from a complex including a Wnt and asFRP, the Wnt may preferably be isolated without using a detergent.

In some embodiments, including, for example, when the Wnt is isolatedbefore forming a complex including a Wnt and a sFRP, isolating the Wntmay include using a detergent. The detergent may include, for example,CHAPS. In some embodiments, the method may further include removing thedetergent from the complex including the Wnt and the sFRP.

In some embodiments, isolating the Wnt may include an aqueouspurification procedure. In some embodiments, isolating the Wnt mayinclude chromatographic separation. Chromatographic separation mayinclude, for example, separation using a SEPHAROSE column.

In some embodiments, the method may include producing and/or expressingat least one of the Wnt and the sFRP. In some embodiments, the Wntand/or the sFRP may be overexpressed. The Wnt and/or the sFRP may beexpressed in mammalian cells, yeast, bacteria, insect cells (S2, Sf9,Sf21), or other cells under the control of appropriate promoters.Cell-free translation systems may also be employed to produce the Wntand/or the sFRP using RNAs. Appropriate cloning and expression vectorsfor use with prokaryotic and eukaryotic hosts are described by, forexample, Sambrook, et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor, N.Y. (1989).

In some embodiments, forming a complex including a Wnt and a sFRP mayinclude co-culturing a Wnt-expressing cell with a sFRP expressing cell.In some embodiments, forming a complex including a Wnt and a sFRP mayinclude co-culturing a Wnt-expressing cell with a sFRP-expressing cell.

In some embodiments, the method may include isolating conditioned mediafrom a cell expressing the Wnt and the sFRP or from a co-culture of aWnt-expressing cell with a sFRP expressing cell. In some embodiments,the method may further include isolating a complex including a Wnt and asFRP from the conditioned media.

Methods of Using

In another aspect, this disclosure describes methods of using anisolated protein complex, as described herein. For example, the isolatedprotein complexes, isolated Wnt, and/or compositions described hereincould be used as a media additive. In some embodiments, the mediaadditive could be used in cell culture including, for example, a stemcell culture or an organoid culture. In some embodiments, the isolatedprotein complexes, isolated Wnt, and/or compositions described hereincould be used in a disease model. In some embodiments, the isolatedprotein complexes, isolated Wnt, and/or compositions described hereincould be used to direct differentiation of cells including, for example,embryonic stem cells or induced pluripotent stem cells. A cell may bedifferentiated to any desirable cell type including, for example, aneuron, neuroectoderm, a cardiac myocyte, a mesenchymal stem cell,and/or a mesendoderm derivative (see, e.g., Lam et al., 2014, SeminNephrol, 34(4):445-461; Spence et al., 2011, Nature, 470:105-109; Paigeet al., 2010, PLoS One 5(6):e11134. doi: 10.1371/journal.pone.0011134;Murashov et al., 2004 FASEB 19(2):252-4).

Exemplary Embodiments Isolated Protein Complex Embodiments

-   -   1. An isolated protein complex comprising a Wnt and a sFRP.    -   2. The isolated protein complex of Embodiment 1, wherein the Wnt        comprises at least one of Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4,        Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b,        Wnt10a, Wnt10b, Wnt11, and Wnt16.    -   3. The isolated protein complex of any one of the previous        Embodiments, wherein the Wnt comprises at least one of Wnt3a,        Wnt5a, Wnt5b, Wnt8a, and Wnt10a.    -   4. The isolated protein complex of any one of the previous        Embodiments, wherein the sFRP comprises at least one of sFRP1,        sFRP2, sFRP3, sFRP4, and sFRP5.    -   5. The isolated protein complex of any one of the previous        Embodiments, wherein the Wnt comprises an active Wnt.    -   6. The isolated protein complex of Embodiment 5, wherein the        active Wnt comprises Wnt that activates β-catenin signaling.    -   7. The isolated protein complex of Embodiment 5 or 6, wherein        the active Wnt comprises Wnt having Wnt reporter activity as        measured using a secreted alkaline phosphatase (SEAP) reporter        assay.    -   8. The isolated protein complex of Embodiment 7, wherein the        isolated protein complex has a 2-fold or greater Wnt reporter        activity than an isolated protein complex that does not comprise        a Wnt, as measured using the SEAP reporter assay.    -   9. The isolated protein complex of any one of the previous        Embodiments, wherein the protein complex is substantially free        of a detergent.    -   10. The isolated protein complex of any one of the previous        Embodiments, wherein the protein complex exhibits an effective        dose of 50 percent (ED₅₀) of less than 1000 ng/mL, less than 500        ng/mL, or less than 100 ng/mL as measured using a HEK293        TCF9-SEAP hFz4/hLRP5 Wnt Reporter assay.    -   11. The isolated protein complex of any one of the previous        Embodiments, wherein the Wnt comprises a mammalian Wnt.    -   12. The isolated protein complex of any one of the previous        Embodiments, wherein the Wnt comprises human Wnt or mouse Wnt.    -   13. The isolated protein complex of any one of the previous        Embodiments, wherein the sFRP comprises a mammalian sFRP.    -   14. The isolated protein complex of any one of the previous        Embodiments, wherein the sFRP comprises human sFRP or mouse        sFRP.    -   15. The isolated protein complex of any one of the previous        Embodiments, wherein the sFRP comprises a tagged sFRP.    -   16. The isolated protein complex of Embodiment 15, wherein the        tagged sFRP comprises histidine-tagged sFRP.    -   17. The isolated protein complex of Embodiment 15 or 16, wherein        the tagged sFRP comprises a C-terminal-tagged sFRP.    -   18. The isolated protein complex of any one of the previous        Embodiments, wherein the Wnt comprises a tagged Wnt.    -   19. The isolated protein complex of Embodiment 18, wherein the        tagged Wnt comprises a histidine-tagged Wnt.    -   20. The isolated protein complex of Embodiment 18 or 19, wherein        the tagged Wnt comprises a C-terminal-tagged Wnt.    -   21. The isolated protein complex of any one of the previous        Embodiments, wherein the complex comprises a Wnt-sFRP fusion        protein.    -   22. The isolated protein complex of Embodiment 21, wherein the        Wnt-sFRP fusion protein comprises a linker.    -   23. The isolated protein complex of Embodiment 22, wherein the        linker comprises a peptide linker.    -   24. The isolated protein complex of any one of the previous        Embodiments, the isolated protein complex further comprises at        least one of R-Spondin 1, R-Spondin 2, R-Spondin 3, and        R-Spondin 4.    -   25. The isolated protein complex of Embodiment 24, wherein the        at least one of R-Spondin 1, R-Spondin 2, R-Spondin 3, and        R-Spondin 4 is fused to the sFRP1, forming a fusion protein.    -   26. The isolated protein complex of Embodiment 25, wherein the        at least one of R-Spondin 1, R-Spondin 2, R-Spondin 3, and        R-Spondin 4 is fused the sFRP1 via a linker.    -   27. The isolated protein complex of Embodiment 26, wherein the        linker comprises a peptide linker.    -   28. The isolated protein complex of any one of Embodiments 25 to        27, the fusion protein further comprising the Wnt.    -   29. The isolated protein complex of Embodiment 24 or Embodiment        28, wherein the at least one of R-Spondin 1, R-Spondin 2,        R-Spondin 3, and R-Spondin 4 is fused to the Wnt, forming a        fusion protein.    -   30. The isolated protein complex of Embodiment 29, wherein the        at least one of R-Spondin 1, R-Spondin 2, R-Spondin 3, and        R-Spondin 4 is fused the Wnt via a linker.    -   31. The isolated protein complex of Embodiment 30, wherein the        linker comprises a peptide linker.    -   32. The isolated protein complex of any one of Embodiments 23,        27, or 31, wherein the peptide linker comprises at least one of        GGGS (SEQ ID NO:1), GGGGS (SEQ ID NO:2), (SEQ ID NO:3), GGGGG        (SEQ ID NO:4), and GSGSGGSGSG (SEQ ID NO:5).    -   33. The isolated protein complex of any one of the previous        Embodiments, the isolated protein complex further comprising        Lipocalin7.    -   34. The isolated protein complex of any one of the previous        Embodiments, the isolated protein complex further comprising        WIF1.    -   35. A method of using the isolated protein complex of any one of        the previous Embodiments.

Wnt Composition Embodiments

-   -   1. A composition comprising a Wnt, wherein the composition is        substantially free of a detergent.    -   2. The composition of Embodiment 1, wherein the Wnt comprises        active Wnt.    -   3. A composition comprising a Wnt, wherein the Wnt comprises        active Wnt.    -   4. The composition of either of Embodiments 2 or 3, wherein the        active Wnt comprises Wnt that activates canonical β-catenin        signaling.    -   5. The composition of either of Embodiments 2 or 3, wherein the        active Wnt comprises Wnt having Wnt reporter activity as        measured using a secreted alkaline phosphatase (SEAP) reporter        assay.    -   6. The composition of Embodiment 5, wherein the composition has        a 2-fold or greater Wnt reporter activity than a composition        that does not comprise Wnt, as measured using the SEAP reporter        assay.    -   7. The composition of any one of Embodiments 2 to 6, wherein the        active Wnt exhibits an effective dose of 50 percent (ED₅₀) of        less than 1000 ng/mL, less than 500 ng/mL, or less than 100        ng/mL as measured using a HEK293 TCF9-SEAP hFz4/hLRP5 Wnt        Reporter assay.    -   8. The composition of any one of the previous Embodiments,        wherein the Wnt comprises at least one of Wnt1, Wnt2, Wnt2b,        Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a,        Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, and Wnt16.    -   9. The composition of any one of the previous Embodiments,        wherein the Wnt comprises at least one of Wnt3a, Wnt5a, Wnt5b,        Wnt8a, and Wnt10a.    -   10. The composition of any one of the previous Embodiments,        wherein the Wnt comprises a mammalian Wnt.    -   11. The composition of any one of the previous Embodiments,        wherein the Wnt comprises human Wnt or mouse Wnt.    -   12. The composition of any one of the previous Embodiments, the        composition further comprising a sFRP.    -   13. The composition of Embodiment 12, wherein the sFRP comprises        at least one of sFRP1, sFRP2, sFRP3, sFRP4, and sFRP5.    -   14. The composition of either of Embodiments 12 or 13, wherein        the composition comprises a Wnt-sFRP fusion protein.    -   15. The composition of Embodiment 14, wherein the Wnt-sFRP        fusion protein comprises a linker.    -   16. The composition of Embodiment 15, wherein the linker        comprises a peptide linker.    -   17. The composition of any one of Embodiments 12 to 15 wherein        the sFRP comprises a mammalian sFRP.    -   18. The composition of any one of Embodiments 12 to 17, wherein        the sFRP comprises human sFRP or mouse sFRP.    -   19. The composition of any one of Embodiments 12 to 18, wherein        the sFRP comprises tagged sFRP.    -   20. The composition of Embodiment 19, wherein the tagged sFRP        comprises histidine-tagged sFRP.    -   21. The composition of Embodiment 20 or 21, wherein the tagged        sFRP comprises a C-terminal-tagged sFRP.    -   22. The composition of any one of the previous Embodiments,        wherein the Wnt comprises tagged Wnt.    -   23. The composition of Embodiment 22, wherein the tagged Wnt        comprises histidine-tagged Wnt.    -   24. The composition of Embodiment 22 or 23, wherein the tagged        Wnt comprises a C-terminal tagged Wnt.    -   25. The composition of any one of the previous Embodiments, the        composition further comprising at least one of R-Spondin 1,        R-Spondin 2, R-Spondin 3, and R-Spondin 4.    -   26. The composition of Embodiment 25, wherein the at least one        of R-Spondin 1, R-Spondin 2, R-Spondin 3, and R-Spondin 4 is        fused to the Wnt, forming a fusion protein.    -   27. The composition of Embodiment 23, wherein the at least one        of R-Spondin 1, R-Spondin 2, R-Spondin 3, and R-Spondin 4 is        fused the Wnt via a peptide linker.    -   28. The composition of any one of Embodiments 25 to 27, the        composition comprising a sFRP, and wherein the at least one of        the Wnt, R-Spondin 1, R-Spondin 2, R-Spondin 3, and R-Spondin 4        is fused to the sFRP, forming a fusion protein.    -   29. The composition of Embodiment 28, wherein the at least one        of the Wnt, R-Spondin 1, R-Spondin 2, R-Spondin 3, and R-Spondin        4 is fused the sFRP via a peptide linker.    -   30. The composition of Embodiment 16, 27, or 29, wherein the        peptide linker comprises at least one of GGGS (SEQ ID NO:1),        GGGGS (SEQ ID NO:2), (SEQ ID NO:3), GGGGG (SEQ ID NO:4), and        GSGSGGSGSG (SEQ ID NO:5).    -   31. The composition of any one of the previous Embodiments, the        composition further comprising Lipocalin7.    -   32. The composition of any one of the previous Embodiments, the        composition further comprising WIF1.    -   33. A method of using the composition of any one of the previous        Embodiments.

Cell Embodiments

-   -   1. A cell overexpressing a Wnt and a sFRP.    -   2. The cell of Embodiment 1, wherein the Wnt comprises at least        one of Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6,        Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11,        and Wnt16.    -   3. The cell of any one of the previous Embodiments, wherein the        Wnt comprises at least one of Wnt3a, Wnt5a, Wnt5b, Wnt8a, and        Wnt10a.    -   4. The cell of any one of the previous Embodiments, wherein the        sFRP comprises at least one of sFRP1, sFRP2, sFRP3, sFRP4, and        sFRP5.    -   5. The cell of any one of the previous Embodiments, wherein the        Wnt comprises an active Wnt.    -   6. The cell of Embodiment 5, wherein the active Wnt comprises        Wnt that activates β-catenin signaling.    -   7. The cell of any one of the previous Embodiments, wherein the        Wnt exhibits an effective dose of 50 percent (ED₅₀) of less than        1000 ng/mL, less than 500 ng/mL, or less than 100 ng/mL as        measured using a HEK293 TCF9-SEAP hFz4/hLRP5 Wnt Reporter assay.    -   8. The cell of any one of the previous Embodiments, wherein the        Wnt comprises a mammalian Wnt.    -   9. The cell of any one of the previous Embodiments, wherein the        Wnt comprises human Wnt or mouse Wnt.    -   10. The cell of any one of the previous Embodiments, wherein the        sFRP comprises a mammalian sFRP.    -   11. The cell of any one of the previous Embodiments, wherein the        sFRP comprises human sFRP or mouse sFRP.    -   12. The cell of any one of the previous Embodiments, wherein the        sFRP comprises tagged sFRP.    -   13. The cell of Embodiment 12, wherein the tagged sFRP comprises        histidine-tagged sFRP.    -   14. The cell of Embodiment 12 or 13, wherein the tagged sFRP        comprises C-terminal-tagged sFRP.    -   15. The cell of any one of the previous Embodiments, wherein the        Wnt comprises tagged Wnt.    -   16. The cell of Embodiment 15, wherein the tagged Wnt comprises        histidine-tagged Wnt.    -   17. The cell of Embodiment 15 or 16, wherein the tagged Wnt        comprises C-terminal-tagged Wnt.    -   18. The cell of any one of the previous Embodiments, wherein the        cell expresses a Wnt-sFRP fusion protein.    -   19. The cell of Embodiment 18, wherein the Wnt-sFRP fusion        protein comprises a linker.    -   20. The cell of Embodiment 19, wherein the linker comprises a        peptide linker.    -   21. The cell of any one of the previous Embodiments, the cell        further expressing at least one of R-Spondin 1, R-Spondin 2,        R-Spondin 3, and R-Spondin 4.    -   22. The cell of Embodiment 21, wherein the at least one of        R-Spondin 1, R-Spondin 2, R-Spondin 3, and R-Spondin 4 is fused        to the sFRP1, forming a fusion protein.    -   23. The cell of Embodiment 22, wherein the at least one of        R-Spondin 1, R-Spondin 2, R-Spondin 3, and R-Spondin 4 is fused        the sFRP1 via a linker.    -   24. The cell of Embodiment 23, wherein the linker comprises a        peptide linker.    -   25. The cell of any one of Embodiments 22 to 27, the fusion        protein further comprising the Wnt.    -   26. The cell of Embodiment 21 or Embodiment 25, wherein the at        least one of R-Spondin 1, R-Spondin 2, R-Spondin 3, and        R-Spondin 4 is fused to the Wnt, forming a fusion protein.    -   27. The cell of Embodiment 26, wherein the at least one of        R-Spondin 1, R-Spondin 2, R-Spondin 3, and R-Spondin 4 is fused        the Wnt via a linker.    -   28. The cell of Embodiment 27, wherein the linker comprises a        peptide linker.    -   29. The cell of any one of Embodiments 20, 24, or 28, wherein        the peptide linker comprises at least one of GGGS (SEQ ID NO:1),        GGGGS (SEQ ID NO:2), (SEQ ID NO:3), GGGGG (SEQ ID NO:4), and        GSGSGGSGSG (SEQ ID NO:5).    -   30. A method of using the cell of any one of the previous        Embodiments.

Cell Composition Embodiments

-   -   1. A composition comprising:        -   a cell overexpressing a Wnt; and        -   a cell overexpressing a sFRP.    -   2. The composition of Embodiment 1, wherein the Wnt comprises at        least one of Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b,        Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b,        Wnt11, and Wnt16.    -   3. The composition of any one of the previous Embodiments,        wherein the Wnt comprises at least one of Wnt3a, Wnt5a, Wnt5b,        Wnt8a, and Wnt10a.    -   4. The composition of any one of the previous Embodiments,        wherein the sFRP comprises at least one of sFRP1, sFRP2, sFRP3,        sFRP4, and sFRP5.    -   5. The composition of any one of the previous Embodiments,        wherein the Wnt comprises an active Wnt.    -   6. The composition of Embodiment 5, wherein the active Wnt        comprises Wnt that activates (3-catenin signaling.    -   7. The composition of any one of the previous Embodiments,        wherein the Wnt exhibits an effective dose of 50 percent (ED₅₀)        of less than 1000 ng/mL, less than 500 ng/mL, or less than 100        ng/mL as measured using a HEK293 TCF9-SEAP hFz4/hLRP5 Wnt        Reporter assay.    -   8. The composition of any one of the previous Embodiments,        wherein the Wnt comprises a mammalian Wnt.    -   9. The composition of any one of the previous Embodiments,        wherein the Wnt comprises human Wnt or mouse Wnt.    -   10. The composition of any one of the previous Embodiments,        wherein the sFRP comprises a mammalian sFRP.    -   11. The composition of any one of the previous Embodiments,        wherein the sFRP comprises human sFRP or mouse sFRP.    -   12. The composition of any one of the previous Embodiments,        wherein the sFRP comprises tagged sFRP.    -   13. The composition of Embodiment 12, wherein the tagged sFRP        comprises histidine-tagged sFRP.    -   14. The composition of Embodiment 12 or 13, wherein the tagged        sFRP comprises C-terminal-tagged sFRP.    -   15. The composition of any one of the previous Embodiments,        wherein the Wnt comprises tagged Wnt.    -   16. The composition of Embodiment 15, wherein the tagged Wnt        comprises histidine-tagged Wnt.    -   17. The composition of Embodiment 15 or 16, wherein the tagged        Wnt comprises C-terminal-tagged Wnt.    -   18. The composition of any one of the previous Embodiments, the        composition further comprising a cell expressing at least one of        R-Spondin 1, R-Spondin 2, R-Spondin 3, and R-Spondin 4.    -   19. The composition of Embodiment 18, wherein the at least one        of R-Spondin 1, R-Spondin 2, R-Spondin 3, and R-Spondin 4 is        fused to the sFRP1, forming a fusion protein.    -   20. The composition of Embodiment 19, wherein the at least one        of R-Spondin 1, R-Spondin 2, R-Spondin 3, and R-Spondin 4 is        fused the sFRP1 via a linker.    -   21. The composition of Embodiment 20, wherein the linker        comprises a peptide linker.    -   22. The composition of Embodiment 18, wherein the at least one        of R-Spondin 1, R-Spondin 2, R-Spondin 3, and R-Spondin 4 is        fused to the Wnt, forming a fusion protein.    -   23. The composition of Embodiment 22, wherein the at least one        of R-Spondin 1, R-Spondin 2, R-Spondin 3, and R-Spondin 4 is        fused the Wnt via a linker.    -   24. The composition of Embodiment 23, wherein the linker        comprises a peptide linker.    -   25. The composition of Embodiments 21 or 24, wherein the peptide        linker comprises at least one of GGGS (SEQ ID NO:1), GGGGS (SEQ        ID NO:2), (SEQ ID NO:3), GGGGG (SEQ ID NO:4), and GSGSGGSGSG        (SEQ ID NO:5).    -   26. The composition of any one of the previous Embodiments,        wherein at least one of the cell overexpressing a Wnt and the        cell overexpressing a sFRP is transfected with a plasmid.    -   27. A method of using the composition of any one of the previous        Embodiments.

Method Embodiments

-   -   1. A method comprising:        -   forming a complex comprising a Wnt and a sFRP; and        -   isolating the Wnt.    -   2. The method of any one of the previous Embodiments, wherein        isolating the Wnt comprises an aqueous purification procedure.    -   3. The method of any one of the previous Embodiments, wherein        isolating the Wnt comprises chromatographic separation.    -   4. The method of any one of the previous Embodiments, the method        further comprising overexpressing the Wnt.    -   5. The method of any one of the previous Embodiments, the method        further comprising overexpressing the sFRP.    -   6. The method of any one of the previous Embodiments, wherein        forming a complex comprising a Wnt and a sFRP comprises        co-culturing a Wnt-expressing cell with a sFRP expressing cell.    -   7. The method of any one of the previous Embodiments, wherein        the complex comprising a Wnt and a sFRP is formed after        isolating the Wnt,    -   8. The method of any one of the previous Embodiments, wherein        isolating the Wnt comprises using a detergent.    -   9. The method of any one Embodiments 1 to 7, wherein the complex        comprising a Wnt and a sFRP is formed before isolating the Wnt.    -   10. The method of any one of Embodiment 1 to 7 or 9, wherein        isolating the Wnt does not comprise using a detergent.    -   11. The method of any one of the previous Embodiments, wherein        the Wnt comprises at least one of Wnt1, Wnt2, Wnt2b, Wnt3,        Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b,        Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, and Wnt16.    -   12. The method of any one of the previous Embodiments, wherein        the Wnt comprises at least one of Wnt3a, Wnt5a, Wnt5b, Wnt8a,        and Wnt10a.    -   13. The method of any one of the previous Embodiments, wherein        the sFRP comprises at least one of sFRP1, sFRP2, sFRP3, sFRP4,        and sFRP5.    -   14. The method of any one of the previous Embodiments, wherein        the Wnt comprises a mammalian Wnt.    -   15. The method of any one of the previous Embodiments, wherein        the Wnt comprises human Wnt or mouse Wnt.    -   16. The method of any one of the previous Embodiments, wherein        the sFRP comprises a mammalian sFRP.    -   17. The method of any one of the previous Embodiments, wherein        the sFRP comprises human sFRP or mouse sFRP.    -   18. The method of any one of the previous Embodiments, wherein        the sFRP comprises tagged sFRP.    -   19. The method of Embodiment 18, wherein the tagged sFRP        comprises histidine-tagged sFRP.    -   20. The method of Embodiment 18 or 19, wherein the tagged sFRP        comprises C-terminal-tagged sFRP.    -   21. The method of any one of the previous Embodiments, wherein        the Wnt comprises a tagged Wnt.    -   22. The method of Embodiment 21, wherein the tagged Wnt        comprises a histidine-tagged Wnt.    -   23. The method of Embodiment 21 or 22, wherein the tagged Wnt        comprises a C-terminal-tagged Wnt.    -   24. The method of any one of the previous Embodiments, wherein        the complex further comprises at least one of R-Spondin 1,        R-Spondin 2, R-Spondin 3, R-Spondin 4, Lipocaline7, and WIF1.    -   25. The method of any one of the previous Embodiments, the        method comprising culturing a cell expressing the Wnt.    -   26. The method of any one of the previous Embodiments, the        method comprising culturing a cell expressing the sFRP.    -   27. The method of claim 26, wherein the cell overexpressing the        Wnt and the cell expressing the sFRP are co-cultured.    -   28. The method of any one of the previous Embodiments, the        method comprising culturing a cell expressing the Wnt and the        sFRP.    -   29. The method of any one of Embodiments 25 to 28, the method        further comprising isolating the conditioned media.    -   30. The method of Embodiment 29, the method comprising isolating        the complex comprising the Wnt and the sFRP from the conditioned        media.    -   31. The method of any one of the previous Embodiments, the        method further comprising removing a detergent from the complex        comprising the Wnt and the sFRP.    -   32. The method of any one of the previous Embodiments, the        method further comprising using the complex as a media additive.    -   33. The method of any one of the previous Embodiments, the        method further comprising adding the complex to a stem cell        culture or an organoid culture.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLES Example 1

This Example describes a method of purifying Wnt proteins thateliminates the use of detergents but maintains the activity ofrecombinant Wnt proteins. The method includes co-expressing sFRPs thatbind to Wnts. Without wishing to be bound by theory, it is believed thatthe sFRPs protect the lipid modification of the Wnt proteins from theaqueous environment. The data presented herein also demonstrate thatsFRPs may be used to complex with and enhance the amount of active Wntin the conditioned media of cells producing Wnts.

Methods Cell Culture and Cell Transfection

All cell lines, including CHO, CHO mWnt-1, CHO msFRP-1, CHOmWnt-1/msFRP-1 and CHO mWnt-1/msFRP-1-His, were cultured in IMDM(ThermoFisher Scientific, Waltham, Mass.), 5% FBS (Corning, Corning,N.Y.) with 2 millimolar (mM) L-Glutamine-Penicillin-Streptomycin(Sigma), as well as appropriate selection antibiotics (Puromycin, G418,and/or Hygromycin; all from ThermoFisher Scientific, Waltham, Mass.).Cells were transfected with expression plasmids using Lipofectamine 2000reagent according to the instructions of the manufacture (ThermoFisherScientific, Waltham, Mass.).

To obtain conditioned media for a HEK293 TCF9-SEAP hFz4/hLRP5 WntReporter assay and Western blots, 5×10⁴ cells were seeded in a 12-welldish and incubated at 37° C., 5% CO₂ in an incubator for four days. Theconditioned media (that is the supernatants) were collected and the celldebris was removed by centrifugation.

HEK293 TCF9-SEAP hFz4/hLRP5 Wnt Reporter Assay

Clonal HEK293 cells expressing full length human LRP5, full length humanFZ4, and 9×T-Cell Factor DNA binding sites (TCF9) upstream of secretedalkaline phosphatase (SEAP) were seeded in 96-well plate and incubatedovernight at 37° C.+5% CO₂. Then the cells were treated with eitherconditioned media or protein for 18 hours. After heat inactivation ofendogenous alkaline phosphatase, 10 microliters (μL) of conditionedmedium from each well was mixed with 50 μL SEAP Reporter AssayBuffer/Substrate (0.1 M Tris-HCl, pH 9.0, 0.1 mM DiFMUP (ThermoFisherScientific, Waltham, Mass.)) and incubated in dark at room temperaturefor 15 minutes to 30 minutes. SEAP activity was measured with excitationat 350 nanometers (nm), emission at 450 nm, and 435 nm cut-off on amicroplate reader (Spectra Max Gemini EM, Molecular Devices, LLC,Sunnyvale, Calif.).

Western Blot Analysis

Conditioned media or recombinant proteins were lysed in 2× reducingsample buffer (20 mM dithiothreitol, 6% SDS, 0.25 molar (M) Tris, pH6.8, 10% glycerol, 10 mM NaF and bromophenyl blue) denatured at 95° C.for 3 minutes and resolved on 4-20% SDS-PAGE gels. The gels weretransferred to PVDF membranes (Millipore, Billerica, Mass.) andincubated with primary antibody in blocking buffer (25 mM Tris, pH 7.4,0.15 M NaCl, 0.1% Tween-20) containing 5% nonfat dry milk at 4° C.overnight. After extensive washings, the membranes were incubated withsecondary antibody in blocking buffer at room temperature for 1 hour.The immunolabeling was revealed by a chemiluminescence reaction usingthe SuperSignal West Pico Chemiluminescent Substrate (ThermoFisherScientific, Waltham, Mass.). Antibodies used for Western blotting wereas follows; Gt×mWnt-1, 1 μg/mL (Catalog No. AF1620, Bio-Techne,Minneapolis, Minn.). Gt x hsFRP-1, 1 μg/mL (Catalog No. AF1384,Bio-Techne, Minneapolis, Minn.). Dk×Gt IgG, HRP, 1:1000 dilution(Catalog No. HAF109, Bio-Techne, Minneapolis, Minn.).

Immunoprecipitation of mWnt1 and msFRP1

5×10⁴ of CHO mWnt-1/msFRP1 cells were seeded in a 12-well dish andincubated at 37° C., 5% CO₂ in an incubator for four days. Theconditioned media were collected and the cell debris removed bycentrifugation. 500 μL of conditioned media were first incubated withimmunoprecipitation antibody for 2 hours to 4 hours at 4° C., and then50μ.L of protein G-agarose beads (Pierce Protein Biology/ThermoFisherScientific, Waltham, Mass.) was added and incubated for 2 hours. Thebound immune complexes were recovered and washed three times inDulbecco's phosphate-buffered saline (DPBS). Proteins were resolved bySDS-PAGE, transferred to PVDF membrane for Western blot analysis.Antibodies used for immunoprecipitations and Western blotting were asfollows; Gt×mWnt-1, 1 μg/mL (Catalog No. AF1620, Bio-Techne,Minneapolis, Minn.). Ms×His, 1 μg/mL (Catalog No. MAB050R, Bio-Techne,Minneapolis, Minn.). Dk×Gt IgG, HRP, 1:1000 dilution (Catalog No.HAF109, Bio-Techne, Minneapolis, Minn.). Gt×Ms IgG, HRP (Catalog No.HAF007, Bio-Techne, Minneapolis, Minn.).

Immunoprecipitation of CHO mWnt2b/msFRP1 Conditioned Media

5×10⁴ of CHO mWnt2b/msFRP1 cells were seeded in the 12-well dish andincubated at 37° C., 5% CO₂ in an incubator for four days. Theconditioned media were collected and the cell debris removed bycentrifugation. 500 μL of conditioned media were first incubated withimmunoprecipitation antibody for 2 hours to 4 hours at 4° C., and then50 μL of protein G-agarose beads (Pierce Protein Biology/ThermoFisherScientific, Waltham, Mass.) was added and incubated for 2 hours. Thebound immune complexes were recovered and washed three times in DPBS.Proteins were resolved by SDS-PAGE, transferred to PVDF membrane forWestern blot analysis. Antibodies used for immunoprecipitations andWestern blotting were as follows; Gt×mWnt2b, 1 μg/mL (Catalog No.AF3900, Bio-Techne, Minneapolis, Minn.). Gt×hsFRP-1, 1 μg/mL (CatalogNo. AF1384 Bio-Techne, Minneapolis, Minn.). Dk×Gt IgG, HRP, 1:1000dilution (Catalog No. HAF109, Bio-Techne, Minneapolis, Minn.).

Co-Culture of Wnt Expressing Cells with msFRP Expressing Cells

5×10⁴ Wnt expressing cells were seeded in a 12-well dish and incubatedat 37° C., 5% CO₂ incubator for three days. 2×10⁵ of msFRP expressingcells were added to the Wnt expressing cells and co-cultured for oneday. The conditioned media were collected and applied to HEK293 Wntreporter cells.

CHO mWnt-1 Cell Treatment with sFRP-1 Protein

A 12-well plate was seeded with 4×10⁵ CHO mWnt-1 cells in 0.6 milliliter(mL) culture media. The cells were incubated at 37° C. for 1 hour andthen added msFRP-1 protein treatment, starting from 50 μg/mL and serialdiluted at 1:2. After 24 hours the conditioned media was collected bybeing centrifuged for 5 minutes to remove cell debris before being addedthe reporter cells.

Purification of Mouse Wnt-1/Mouse sFRP Complex

The conditioned media from CHO cells that co-expressed mouse Wnt-1 andmouse sFRP1 were loaded onto a SP sepharose Fast Flow column (GEHealthcare, Chicago, Ill.) equilibrated with 20 mM MOPS, 0.1 M NaCl, pH6.8. A linear gradient of high salt buffer (20 mM MOPS, 1.6 M NaCl, pH6.8) was applied to elute the bound proteins. The SDS-PAGE with silverstaining was used to monitor elution of mouse sFRP1 (˜37 kDa), while theWestern blot probed with anti-mouse Wnt-1 was used to monitor elution ofmouse Wnt-1 (˜40 kDa). The protein peak that included both Wnt-1 andsFRP1 was collected. After concentrating, the pooled peak was loadedonto a Superdex-200 column (GE Healthcare, Chicago, Ill.) to separateWnt-1/sFRP1 complex from free sFRP1, monitored by both SDS-PAGE silverstaining and Western blot as in the previous step.

Wnt3a/sFRP ELISA Binding Assays

A 96-well tissue culture plate was blocked with 1% BSA in PBS and usedas an in-solution binding plate. 120 μL of total volume containing 100ng/mL recombinant mouse (rm) Wnt3a biotinylated protein and sFRPs (1:3serial dilution) were incubated overnight at 4° C. in the binding plate.Then 100 μL of binding solution was transferred to streptavidin coated96-well ELISA plate, incubating overnight at 4° C. followed by the HRPdetection. The mouse sFRP1 was detected with a goat anti-mouse sFRP1antibody, mouse sFRP2 and mouse sFRP3 were His-tagged and were detectedwith a mouse anti-His antibody, mouse sFRP4 was detected with a sheepanti-mouse sFRP4 antibody, and the HA-tagged mouse sFRP5 was detectedwith a mouse anti-HA peptide. Anti-HRP secondary antibodies were used todetect the primary antibodies followed by colorimetric readouts.

Results

To determine if expressing mouse sFRP1 (msFRP1) or mouse sFRP1-His(msFRP1-His) would enhance the amount of mouse Wnt1 (mWnt1) in theconditioned media (CM) of CHO cells, stable, clonal lines expressingmWnt1, msFRP1, mWnt1 and msFRP1, and mWnt1 and msFRP1-His were made.Western blot analysis of conditioned media demonstrated thatco-expression of mWnt1 along with msFRP1 or msFRP1-His resulted insignificantly higher levels of mWnt1 in the conditioned media comparedto CHO, CHO mWnt1 or CHO msFRP1 conditioned media (FIG. 1A). CHO cellsoverexpressing msFRP1 also clearly showed higher levels of msFRP1compared to msFRP1 levels in CHO, or CHO mWnt1 conditioned media (FIG.1A).

The same conditioned media used in Western blot analysis of FIG. 1A wasalso tested in a HEK293 Wnt Reporter line that expresses human Frizzled4(hFz4) and human LRP5 (hLRP5) (also referred to herein as conducting anHEK293 hFz4/hLRP5 Wnt reporter assay). Conditioned media from CHO cellsresulted in Wnt reporter activation only when mWnt1 was co-expressedwith msFRP1 or msFRP1-His (FIG. 1B). Wild type CHO, CHO mWnt1, and CHOmsFRP1 conditioned media did not activate the Wnt reporter at a levelbeyond background. These data are consistent with msFRP1 or msFRP1-Hisoverexpression resulting in higher levels of active mWnt1 in theconditioned media of cells expressing both mWnt1 and msFRP1 ormsFRP1-His (FIG. 1).

To test if a Wnt and a sFRP could bind to each other in a complex, theinteraction of mWnt1 and msFRP1 was tested. Immunoprecipitationexperiments demonstrated that mWnt1 and msFRP1-His are bound in acomplex (FIG. 2). Immunoprecipitation with an anti-Wnt1 antibody andblotting with an anti-His antibody resulted in the detection of a bandof 37 kDa, the expected size of msFRP1-His (FIG. 2, lane 2).Immunoprecipitation with an anti-His antibody and blotting with ananti-Wnt1 antibody resulted in the detection of a band of 42 kDa, theexpected size of mWnt1 (FIG. 2, lane 5). These data suggest that mWnt1and msFRP1 are bound to each other in a complex in the conditioned mediaof CHO cells expressing both mWnt1 and msFRP1.

To test if mouse sFRP1 (msFRP1) binds to mWnt2b in CHO conditioned mediaexpressing both mouse Wnt2b (mWnt2b) and msFRP1. Immunoprecipitationexperiments were performed by adding anti-Wnt2b, anti-hsFRP1, or noantibody control to equal amounts of CHO cell conditioned mediaoverexpressing mWnt2b alone or mWnt2b and msFRP1 (Clone 18). FIG. 2Bshows that immunoprecipitation with anti-mWnt2b antibodies followed byblotting with anti-hsFRP1 antibodies resulted in the detection of sFRP1protein at around 35 kDa (arrows in FIG. 2B), demonstrating that mWnt2band msFRP1 are physically interacting in the conditioned media of CHOcells expressing both mWnt2b and msFRP1. This 35 kDa band was notdetected in the conditioned media of CHO cells expressing only mWnt1when immunoprecipitations were performed with anti-mWnt2b and blottedwith anti-hsFRP1.

Further immunoprecipitation experimentation demonstrates that msFRP1does bind to mWnt2b in CHO conditioned media expressing both mWnt2b andmsFRP1. Immunoprecipitation experiments were performed by addinganti-Wnt2b, anti-hsFRP1, or no antibody control to equal amounts of CHOcell conditioned media overexpressing mWnt2b alone or mWnt2b and msFRP1(Clone 18). FIG. 2C shows that immunoprecipitation with anti-hsFRP1antibodies followed by blotting with anti-mWnt2b antibodies resulted inthe detection of mWnt2b protein at around 42 kDa (arrow in FIG. 2C),demonstrating that mWnt2b and msFRP1 are physically interacting in theconditioned media of CHO cells expressing both mWnt2b and msFRP1. This42 kDa mWnt1 band was not detected in the conditioned media of CHO cellsexpressing only mWnt1 when immunoprecipitations were performed withanti-hsFRP1 and blotted with anti-mWnt1.

Further experiments were designed to understand why an increase inlevels of active mWnt1 was observed in the conditioned media of cellsexpressing both mWnt1 and msFRP1. When CHO cells expressing only mWnt1were co-cultured with HEK293 hFz4/hLRP5 Wnt reporter cells (alsoreferred to herein as HEK293 Wnt reporter cells), robust activation ofWnt reporter activity is detected, demonstrating that CHO cellsexpressing mWnt1 make active mWnt1 protein (Table 1, row 3). When theconditioned media from CHO mWnt1 cells is added to HEK293 Wnt reportercells, no Wnt reporter activation is detected (Table 1, row 4). Thesedata suggest that the active mWnt1 protein is not in the conditionedmedia, but rather is localized on the cell surface of the CHO mWnt1cells. When CHO cells expressing msFRP1 or msFRP5 alone are co-culturedwith HEK293 Wnt reporter cells, no activity was detected (Table 1, row5). In addition, when the conditioned media from CHO cells expressingmsFRP1 or msFRP5 was added to HEK293 Wnt reporter cells, no activity wasdetected (Table 1, row 6), suggesting that sFRP1 or sFRP5 are notcapable of enhancing Wnt signaling without co-expression of mWnt1. Whenthe conditioned media of CHO mWnt1 and the conditioned media of CHOmsFRP1 or msFRP5 were combined together before adding to the HEK293 Wntreporter, no activity was detected (Table 1, row 7). When theconditioned media of CHO mWnt1 cells was added to CHO msFRP1 or msFRP5expressing cells and cultured overnight prior to adding the conditionedmedia to HEK293 Wnt reporter cells, no activity was detected (Table 1,row 8). When CHO msFRP1 or msFRP5 conditioned media was added to CHOmWnt1 expressing cells overnight followed by adding this conditionedmedia to HEK293 Wnt reporter cells, reporter activity was detected(Table 1, row 9). When recombinant hsFRP1 or hsFRP5 protein was added toCHO mWnt1 expressing cells overnight, followed by adding thisconditioned media to HEK293 Wnt reporter cells, activation of the Wntreporter was observed (Table 1, row 11). Finally, if both mWnt1 andmsFRP1 or msFRP5 were co-expressed in the same CHO cells, theconditioned media activated the HEK293 Wnt reporter (Table 1, row 10).

TABLE 1 Reporter Treatment Activity 1 CHO cells (co-cultured withreporter cells) − 2 CHO cell conditioned media (CM) − 3 CHO mWnt1cells + 4 CHO mWnt1 cell CM − 5 CHO mFRP1 or msFRP5 cells − 6 CHO mFRP1or msFRP5 cell CM − 7 CHO mWnt1 CM + CHO sFRP1/5 CM − 8 CHO mWnt1 CM +CHO sFRP1/5 Cell − 9 CHO mWnt1 cell cultured with CHO sFRP1/5 CMovernight, + then adding this CM to the Wnt reporter cells 10 CHOmWnt1/sFRP1/5 CM (Stable line expressing both + mWnt1 and sFRP1 or sFRP5in the same cell) 11 Adding sFRP1/5 protein to CHO mWnt1 expressingcells + overnight and then adding this CM to Wnt reporter cells.

sFRPs have been observed to act both as positive and negative regulatorsof Wnt activity. Initially, several publications demonstrated sFRPinhibition of Wnt activity (Leyns et al. Cell 88:747-756 (1997); Wang etal. Cell 88:757-766 (1997)), but later studies showed that sFRPs canpotentiate Wnt signaling at physiological doses of sFRPs (Mii et al.Development 136:4083-4088 (2009); Holly et al. Dev. Biol. 388:192-204(2014)). To test for this biphasic activity of sFRPs, a dose series ofrecombinant sFRP1 protein were added to CHO mWnt1 expressing cells for24 hours followed by removal of this conditioned media to treat HEK293Wnt reporter cells. A bell-shaped curve was observed with relativelylower doses of sFRP resulting in enhancement of mWnt1 activity andhigher concentrations of sFRP1 resulting in a return of Wnt signalingback to baseline levels (FIG. 3).

CHO mWnt1/msFRP1 expressing cells were used to purify a mWnt1/msFRP1protein complex. CHO mWnt1/msFRP1 expressing cells were transitionedfrom 5% FBS containing media to 2% FBS containing media so they could begrown in suspension. After 9 days in culture, the CHO mWnt1/msFRP1conditioned media was isolated and the mWnt1/msFRP1 complex was purifiedby an ion exchange chromatography (FIG. 4 A-C) followed by gelfiltration chromatography (FIG. 4 D-E). sFRP1 tightly binds to a cationexchanger SP sepharose column, and a NaCl concentration of greater than1 molar (M) is required to elute the protein, while Wnt-1 is usuallyeluted with NaCl concentration less than 0.3 M. If sFRP and Wnt1 did notform a complex, sFRP1 and Wnt-1 should be eluted at different peaks.However, the Western blot probed with anti-Wnt-1 antibody (FIG. 4B)showed that Wnt-1 was co-eluted with sFRP1 (FIG. 4C) in a later peakfrom SP sepharose column (FIG. 4A), suggesting complex formation betweensFRP1 and Wnt-1. sFRP1 was detected with Silver staining, but Wnt-1 washardly visible, indicating a large amount of free sFRP1 was present.Because of the size difference between sFRP1/Wnt-1 complex and freesFRP, gel filtration chromatography successfully separated these twodifferent populations. The sFRP1/Wnt-1 complex was eluted in the earlierpeak, while the free sFRP1 was eluted in the later peak (FIG. 4D). BothsFRP1 and Wnt-1 were detected in the complex (earlier) peak withCoomassie blue staining on SDS-PAGE (FIG. 4E). The purified sFRP1/Wnt-1complex was loaded onto SDS-PAGE and stained with Coomassie Blue. Twobands, at 37 kDa and 52 kDa, were observed, with roughly 1:1 molar ratiobased on densitometry analysis (FIG. 4F). Characterization of theprotein complex by Western blotting detected both mWnt1 and msFRP1 inthe purified complex sample (FIG. 5). N-terminal sequencing of purifiedcomplex identified both mouse Wnt-1 and mouse sFRP-1 sequence withoutany other protein sequence detected. Binding of mWnt1 and msFRP1 inconditioned media suggests that mWnt1 and msFRP1 are likely associatedin a complex when purified.

The recombinant mWnt1/msFRP1 purified protein was tested for activity ina HEK293 Wnt reporter assay. Treatment of HEK293 Wnt reporter cells withrecombinant msFRP1 protein did not result in activation of the Wntreporter above untreated background levels (FIG. 6B). Treatment ofHEK293 Wnt reporter cells with the mWnt1/msFRP1 protein complex didresult in a robust activation of the Wnt reporter (FIG. 6A, FIG. 6B). Asadditional purification steps were added to the mWnt1/msFRP1purification procedure (FIG. 4), a mWnt1/msFRP1 complex that was evenmore potent compared to the currently available most potent purified Wntin CHAPS buffer (Wnt3a) was obtained (FIG. 6D).

These data demonstrate that sFRPs can increase the amount of activemWnt1 protein in conditioned media when sFRPs interact with CHO cellsmaking mWnt1. To address whether sFRP-induced liberation of Wnt wasspecific for mWnt1 or whether could sFRPs could work in a similarfashion with other Wnt family members, additional sFRP co-cultureexperiments were performed with mWnt1, mWnt2b, and human Wnt6 (hWnt6).

When CHO cells expressing msFRP1 were co-cultured with CHO cellsexpressing mWnt2b and the resulting conditioned media was added toHEK293 Wnt reporter cells, activation of the Wnt reporter was detected(FIG. 7B). Interestingly, co-cultures of CHO mWnt2b cells with CHOmsFRP5 did not result in Wnt activity in the conditioned media (FIG. 7B)similar to the conditioned media from co-cultures of CHO mWnt1 and CHOmsFRP5 cells (FIG. 7A). Conditioned media from HEK293 expressing hWnt6co-cultured with CHO msFRP1 and msFRP5 activated HEK293 Wnt reportercells (FIG. 7C) similar to what was seen with CHO mWnt1 co-cultureexperiments with CHO sFRP1 and sFRP5 (FIG. 7A). These data demonstratethat sFRPs can enhance the liberation of not only mWnt1 from the cellsurface of mWnt1 expressing cells, but sFRPs can also liberate mWnt2band hWnt6 from the cell surface of cells expressing mWnt2b or hWnt6.These data also suggest that sFRPs can be used to free many, if not all,Wnt proteins bound to the cell membrane to facilitate the purificationactive Wnt/sFRP complexes.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forinstance, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference. In the event that anyinconsistency exists between the disclosure of the present applicationand the disclosure(s) of any document incorporated herein by reference,the disclosure of the present application shall govern. The foregoingdetailed description and examples have been given for clarity ofunderstanding only. No unnecessary limitations are to be understoodtherefrom. The invention is not limited to the exact details shown anddescribed, for variations obvious to one skilled in the art will beincluded within the invention defined by the claims.

What is claimed is:
 1. An isolated protein complex comprising a Wnt anda sFRP.
 2. The isolated protein complex of claim 1, wherein the Wntcomprises one or more of Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a,Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b,Wnt11, and Wnt16.
 3. The isolated protein complex of claim 1, whereinthe sFRP comprises one or more of sFRP1, sFRP2, sFRP3, sFRP4, and sFRP5.4. The isolated protein complex of claim 1, wherein the Wnt comprises anactive Wnt.
 5. The isolated protein complex of claim 4, wherein theactive Wnt comprises Wnt having Wnt reporter activity as measured usinga secreted alkaline phosphatase (SEAP) reporter assay.
 6. The isolatedprotein complex of claim 1, wherein the protein complex is substantiallyfree of a detergent.
 7. The isolated protein complex of claim 1, whereinthe Wnt comprises a mouse Wnt and the sFRP comprises a mouse sFRP. 8.The isolated protein complex of claim 1, wherein the Wnt comprises amouse Wnt1 and the sFRP comprises a mouse sFRP1.
 9. The isolated proteincomplex of claim 1, wherein the protein complex exhibits an effectivedose of 50 percent (ED₅₀) of less than 500 ng/mL, as measured using aHEK293 TCF9-SEAP hFz4/hLRP5 Wnt Reporter assay.
 10. A compositioncomprising a Wnt, wherein the composition is substantially free of adetergent.
 11. The composition of claim 10, wherein the Wnt comprisesone or more of Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6,Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, andWnt16.
 12. The composition of claim 10, wherein the Wnt comprises activeWnt.
 13. The composition of claim 12, wherein the active Wnt comprisesWnt having Wnt reporter activity as measured using a secreted alkalinephosphatase (SEAP) reporter assay.
 14. The composition of claim 10,wherein the composition further comprises a sFRP.
 15. The composition ofclaim 14, wherein the sFRP comprises one or more of sFRP1, sFRP2, sFRP3,sFRP4, and sFRP5.
 16. The composition of claim 14, wherein the Wntcomprises a mouse Wnt and the sFRP comprises a mouse sFRP.
 17. Thecomposition of claim 14, wherein the Wnt comprises a mouse Wnt1 and thesFRP comprises a mouse sFRP1.
 18. The composition of claim 10, whereinthe Wnt exhibits an effective dose of 50 percent (ED₅₀) of less than 100ng/mL, as measured using a HEK293 TCF9-SEAP hFz4/hLRP5 Wnt Reporterassay.
 19. The composition of claim 10, the composition furthercomprising one or more of R-Spondin 1, R-Spondin 2, R-Spondin 3,R-Spondin 4, Lipocalin7, or WIF1.
 20. A method comprising:overexpressing a Wnt1 and an sFRP1; forming a complex comprising theWnt1 and the sFRP1; and isolating the Wnt1, wherein isolating the Wnt1comprises an aqueous purification procedure and wherein isolating theWnt does not comprise using a detergent.